PROJECT 2 ABSTRACT PI: Daniel J. Rader, M.D. Recent evidence supports the potential of adeno-associated virus (AAV)-mediated liver-directed gene therapy as an effective therapeutic intervention for a variety of metabolic disorders. Monogenic lipid disorders are excellent models for the development of liver-directed gene therapy. Based on previous work funded by this P01 we are now poised to use a first-generation AAV8 clinical candidate to launch a clinical trial (Project 1) for one such disorder, Homozygous Familial Hypercholesterolemia (hoFH). This lipid disorder is caused by loss- of-function mutations in the LDL receptor (LDLR) and is associated with severe hypercholesterolemia and markedly premature atherosclerotic cardiovascular disease (ASCVD). In this Project 2, we propose to expand on our preclinical proof-of-concept studies for the treatment of hoFH by developing a second-generation AAV clinical candidate vector expressing an engineered LDLR transgene of superior efficacy for the correction of hoFH. Another serious lipid disorder is Familial LCAT Deficiency (FLD), which results in accumulation of unesterified cholesterol and chronic kidney disease progressing to end-stage renal failure. In contrast to the LDL receptor, LCAT is a secreted protein and restoration of a small fraction of the normal levels in blood would be expected to ameliorate the disease and prevent progressive renal disease. In this Project 2, we will develop an optimized clinical candidate vector for the treatment of FLD. Our goal is to optimize the hLDLR and hLCAT transgenes and work closely with Project 3 to optimize AAV-based vectors for correction of hoFH and FLD, such that the minimum efficacious dose of the clinical candidate vector is as low as possible. The overall intent of Project 2 is to lay the preclinical groundwork for conducting Phase 1 clinical trials using a second generation AAV-hLDLR for treatment of hoFH and the first AAV8-hLCAT for the treatment of FLD.